Liver Mitochondria Proteomics: Protein and PTM

Liver Mitochondria Proteomics:
Protein and PTM Quantitation
Jenny Ho,1 Loïc Dayon, 2 John Corthésy, 2 Umberto De Marchi, 2 Antonio Núñez, 2 Rosa Viner, 3
Michael Blank, 3 Steve Danielson, 3 Madalina Oppermann,1 Martin Hornshaw,1
Martin Kussmann, 2,4,5 Andreas Wiederkehr2
1
Thermo Fisher Scientific, Hemel Hempstead, UK; 2Nestlé Institute of Health Sciences, Lausanne,
Switzerland; 3Thermo Fisher Scientific, San Jose, CA, USA; 4Faculty of Life Sciences,
Ecole Polytechnique Fédérale Lausanne (EPFL), Lausanne, Switzerland;
5
Faculty of Sciences, Aarhus University, Aarhus, Denmark
Overview
Results
Methods: Relative quantitation of proteins using TMT 10-plex and LC/MS3.
The TMT 10-plex reagents util
isotopes; therefore, to baseline
m/z 200) were acquired (Figur
Purpose: Towards the understanding of biological aging by deep differential profiling
of the liver mitochondrial proteome.
Results: Quantitative changes of the liver mitochondrial proteome were observed in
different age groups.
Introduction
Identification and Quantitati
FIGURE 2. TMT 10-plex reag
spectra acquired at 60,000 r
To characterize changes in the mitochondrial proteome associated with the process of
aging, liver mitochondria from different age groups of rats were studied. The liver
proteomes from three age groups of rats – young (8 month old), middle (18 month
old), and old (24 month old) – were labeled with Tandem Mass Tags (TMT)1, and
analyzed using nanoLC-MSn workflows to maximize both qualitative and quantitative
information on the mitochondrial proteome.
Methods
Sample Preparation
Liver tissues were homogenized and mitochondria were isolated by differential
centrifugation2. Proteins were then reduced, alkylated, and precipitated with
methanol/chloroform/water. Mitochondria proteins were re-dissolved and digested with
trypsin. Samples were then labeled with Thermo Scientific™ TMT 10-plex™ reagents
according to the manufacturer’s instructions. The experimental strategy consisted of
three age groups, young, middle, and old animals with six biological replicates per age
group. The biological replicates were divided into two experiments, referred to as Exp
A and Exp B. Each experiment consisted of 9 samples labeled with TMT channel 126130 and a pooled age group sample labeled with TMT channel 131. Aliquots from
each sample were mixed in equimolar ratios. The experimental strategy is summarized
in Figure 1.
FIGURE 1. Experimental design
Co-isolation interference has
accuracy when using isobaric
to address co-isolation interfe
precursor was fragmented by
precursor selection of multipl
HCD MS3 (Figure 3). Ion trap
Orbitrap MS3 spectra contain
quantitation. Figure 4 shows
MS3 approach compared to t
FIGURE 3. MS3 approach e
TMT-labelled peptides
LC-MSn
All samples were analyzed by nanoLC coupled to the Thermo Scientific™ Orbitrap
Fusion™ Tribrid™ mass spectrometer. Peptides were separated using a Thermo
Scientific™ Acclaim™ PepMap™ C18 column, 50 cm x 75 µm ID, 3 µm, employing a
water/acetonitrile/0.1% formic acid gradient from 5-35% over 360 minutes. TMTlabeled peptides were analyzed by data-dependent top speed using an MS3
approach. During a maximum 3 s cycle time, the most abundant multiply charged
parent ions were selected for CID MS2 in the ion trap followed by Synchronous
Precursor Selection (SPS) of up to 10 parent ions from the MS2 for HCD MS3. MS3
spectra were acquired at 60,000 resolution (at m/z 200) in the Orbitrap MS. All
samples were analyzed in triplicate.
Data Analysis
For peptide and protein identification as well as TMT quantitation, data were
processed using Thermo Scientific™ Proteome Discoverer™ software version 1.4.
Spectra were searched against a UniProt rat database using the SEQUEST® HT
search engine. Static modifications included carbamidomethylation (C) and
TMTsixplex (N-terminal). Dynamic modifications included oxidation (M), TMTsixplex
(K), acetylation (K), and phosphorylation (S,T,Y). Resulting peptide hits were filtered
for maximum 1% FDR using the Percolator algorithm. The TMT 10-plex quantification
method within Proteome Discoverer software was used to calculate the reporter
ratios with mass tolerance ±10ppm without applying isotopic correction factors. Only
peptide spectra containing all reporter ions were designated as “quantifiable spectra”.
A protein ratio is expressed as a median value of the ratios for all quantifiable spectra
of unique peptides derived from the protein. The MS3 approach generated CID MS2
spectra for identification and HCD MS3 for quantitation.
2 Liver Mitochondria Proteomics: Protein and PTM Quantitation
FIGURE 4. Orbitrap Fusion
for MS3 and MS2 approache
10--plex labelled peptides
g by deep differential profiling
0-plex and LC/MS3.
proteome were observed in
Results
Identification and Quantification o
Identification and Quantitation of TMT-labeled Peptides Using the MS3 Approach
The TMT 10-plex reagents utilize the 6 mDa mass difference between 13C and 15N
isotopes; therefore, to baseline resolve all ten, reporter ions >60,000 resolution (at
m/z 200) were acquired (Figure 2).
FIGURE 2. TMT 10-plex reagents: structures, reporter ion masses, and mass
spectra acquired at 60,000 resolution (at m/z 200)
TMT-labeled peptides from Exp A an
using a 360 min gradient. Each sam
loaded on column. Representative b
shown in Figure 5.
FIGURE 5. Representative base p
associated with the process of
ts were studied. The liver
nth old), middle (18 month
m Mass Tags (TMT)1, and
h qualitative and quantitative
isolated by differential
and precipitated with
re-dissolved and digested with
ific™ TMT 10-plex™ reagents
mental strategy consisted of
ix biological replicates per age
xperiments, referred to as Exp
labeled with TMT channel 126hannel 131. Aliquots from
mental strategy is summarized
Thermo Scientific™ Orbitrap
separated using a Thermo
x 75 µm ID, 3 µm, employing a
% over 360 minutes. TMTp speed using an MS3
abundant multiply charged
ollowed by Synchronous
the MS2 for HCD MS3. MS3
) in the Orbitrap MS. All
Co-isolation interference has been shown to contribute to a decrease in quantitative
accuracy when using isobaric tags such as TMT3,4. An MS3 approach was developed
to address co-isolation interference on the Orbitrap Fusion Tribrid MS, where each
precursor was fragmented by CID MS2 followed by HCD MS3. The synchronous
precursor selection of multiple MS2 ions using the ion trap subsequently underwent
HCD MS3 (Figure 3). Ion trap MS2 spectra were used for peptide identification, while
Orbitrap MS3 spectra containing the TMT 10-plex reporter ions were used for
quantitation. Figure 4 shows acquisition workflows and instrument settings for the
MS3 approach compared to the MS2 approach on the Orbitrap Fusion MS.
FIGURE 3. MS3 approach employing SPS for identification and quantification of
TMT-labelled peptides
FIGURE 4. Orbitrap Fusion MS acquisition workflows and instrument settings
for MS3 and MS2 approaches for the identification and quantification of TMT
10--plex labelled peptides
Each group was analyzed in triplica
performed, resulting in approximate
peptides at 1% FDR (Percolator alg
identified when all six data files were
In addition, 84% of the identified pro
ions were present), and 817 mitocho
quantified using ≥2 unique peptides
The number of peptides identified u
Exp A was acquired using the instru
LC gradient. Figure 7 summarizes th
where approximately 12% less prote
MS2 approach using the same grad
FIGURE 6. Summary of the numb
and quantified by LC-MS3 using 3
FIGURE 7. Summary of the numb
using MS2 compared to the MS3 a
analytical experiments)
uantitation, data were
erer™ software version 1.4.
using the SEQUEST® HT
omethylation (C) and
ed oxidation (M), TMTsixplex
lting peptide hits were filtered
The TMT 10-plex quantification
d to calculate the reporter
otopic correction factors. Only
nated as “quantifiable spectra”.
atios for all quantifiable spectra
approach generated CID MS2
.
Thermo Scientific Poster Note • PN-64105-ASMS-EN-0614S 3
Identification and Quantification of the Mitochondrial Proteome
Peptides Using the MS3 Approach
difference between 13C and 15N
orter ions >60,000 resolution (at
eporter ion masses, and mass
00)
Quantitative Changes to the
nanoLC-MS3
TMT-labeled peptides from Exp A and B (Figure 1) were analyzed by
using a 360 min gradient. Each sample was analyzed in triplicate and 2 µg were
loaded on column. Representative base peak chromatograms for Exp A and B are
shown in Figure 5.
FIGURE 5. Representative base peak chromatograms for groups A and B
To study changes to the mitoch
groups were compared using is
concurrent identification and qu
in one LC-MS3 experiment.3 Th
age were used, in which six bio
replicates were divided into two
(Figure 1). Each group consiste
and labeled using TMT channe
pooled age group sample.
All samples were normalized to
channel 131). Figure 8 summa
samples for selected mitochond
same age group were observed
abundance with increasing age
shows box plots summarizing t
proteins for different age group
FIGURE 8. Normalized ratios
proteins in all biological sam
ribute to a decrease in quantitative
An MS3 approach was developed
ap Fusion Tribrid MS, where each
by HCD MS3. The synchronous
e ion trap subsequently underwent
used for peptide identification, while
reporter ions were used for
s and instrument settings for the
n the Orbitrap Fusion MS.
,4.
dentification and quantification of
Each group was analyzed in triplicate; thus six analytical experiments were
performed, resulting in approximately two days of analysis time. A total of 15,464
peptides at 1% FDR (Percolator algorithm) and 2,635 protein groups were
identified when all six data files were combined for the database search (Figure 6).
In addition, 84% of the identified protein groups were quantified (where all reporter
ions were present), and 817 mitochondrial protein groups were identified and 631
quantified using ≥2 unique peptides when all six data files were combined.
The number of peptides identified using MS3 was compared to the MS2 approach.
Exp A was acquired using the instrument settings shown in Figure 4 and a 180 min
LC gradient. Figure 7 summarizes the number of peptides and proteins identified,
where approximately 12% less proteins were identified in the MS3 compared to the
MS2 approach using the same gradient.
FIGURE 6. Summary of the number of peptides and protein groups identified
and quantified by LC-MS3 using 360 min gradient (n=6 analytical experiments)
FIGURE 9. Box plots summa
age groups.
rkflows and instrument settings
tion and quantification of TMT
FIGURE 7. Summary of the number of peptides and protein groups identified
using MS2 compared to the MS3 approach using a 180 min gradient (n=2
analytical experiments)
4 Liver Mitochondria Proteomics: Protein and PTM Quantitation
Proteome
nanoLC-MS3
analyzed by
riplicate and 2 µg were
rams for Exp A and B are
s for groups A and B
Quantitative Changes to the Mitochondrial Proteome upon Aging
Profiling Quantitative Changes to the
To study changes to the mitochondrial proteome upon aging, rats from different age
groups were compared using isobaric labeling and LC-MS3. TMT10-plex enables
concurrent identification and quantification of proteins from up to 10 different samples
in one LC-MS3 experiment.3 Three age groups consisting of young, middle, and old
age were used, in which six biological replicates were used per age group. Biological
replicates were divided into two experiments, referred to as Exp A and Exp B
(Figure 1). Each group consisted of three rats per age group (nine samples in total)
and labeled using TMT channels 126-130, while channel 131 was used to label a
pooled age group sample.
To determine protein abundance distrib
age groups were transformed to a Log2
GProX software5. As shown in Figure 1
which could be further separated into 6
All samples were normalized to the corresponding pooled age group sample (TMT
channel 131). Figure 8 summarizes the calculated normalized ratios for all biological
samples for selected mitochondrial proteins. Although biological variations within the
same age group were observed, there was a subtle trend of increasing protein
abundance with increasing age. This trend can be clearly observed in Figure 9, which
shows box plots summarizing the distribution of protein ratio for all mitochondrial
proteins for different age groups.
.
FIGURE 8. Normalized ratios (against pooled age group) for four mitochondrial
proteins in all biological samples
experiments were
is time. A total of 15,464
otein groups were
atabase search (Figure 6).
antified (where all reporter
s were identified and 631
s were combined.
Conclusion
red to the MS2 approach.
in Figure 4 and a 180 min
s and proteins identified,
n the MS3 compared to the
protein groups identified
6 analytical experiments)
protein groups identified
0 min gradient (n=2
FIGURE 10. Profiling changes to pro
FIGURE 9. Box plots summarizing the distribution of protein ratios for different
age groups.

The multiplexing capabilities of T
samples in one LC-MSn experime

Quantitative changes to the mitoc
using TMT 10-plex and LC-MS3.
levels with increasing age was ob
dissected into different expressio

A total of 35 TMT-labeled acetyla
phosphopeptides were identified
Further experiments include the e
peptides prior to TMT-labeling an
analysis.
References
1. Dayon, L. et al. Relative Quantific
Fluids by MS/MS Using 6-plex Is
2. U. De Marchi et al., Influences of
Monoamine oxidase activity on a
transition, Cellular and Molecular
3. Ting et al. MS3 Eliminates Ratio D
Proteomics. Nature Methods, 201
4. Viner et al. ASMS 2013 poster: In
Quantitation from 6-to 10-Plex Re
5. Rigbolt, K.T. et al., GProX, a Use
and Visualization of Quantitative
Proteomics, 2011, 10(8):O110.00
SEQUEST is a trademark of the University of Wash
Scientific and its subsidiaries.
This information is not intended to encourage use o
intellectual property rights of others.
Thermo Scientific Poster Note • PN-64105-ASMS-EN-0614S 5
e upon Aging
aging, rats from different age
MS3. TMT10-plex enables
om up to 10 different samples
ng of young, middle, and old
sed per age group. Biological
o as Exp A and Exp B
group (nine samples in total)
el 131 was used to label a
ed age group sample (TMT
malized ratios for all biological
iological variations within the
nd of increasing protein
ly observed in Figure 9, which
ratio for all mitochondrial
Profiling Quantitative Changes to the Mitochondrial Proteome upon Aging
To determine protein abundance distribution profiles, the protein ratios from the three
age groups were transformed to a Log2 scale and clustering was performed using
GProX software5. As shown in Figure 10, 41 proteins showed quantitative differences
which could be further separated into 6 different profile clusters.
FIGURE 10. Profiling changes to protein expression levels upon aging
.
roup) for four mitochondrial
Conclusion
of protein ratios for different

The multiplexing capabilities of TMT allows the comparison of up to 10 biological
samples in one LC-MSn experiment.

Quantitative changes to the mitochondria proteome in aging rats were made
using TMT 10-plex and LC-MS3. A subtle trend in increasing protein expression
levels with increasing age was observed. Interestingly, this trend could be further
dissected into different expression profiles.

A total of 35 TMT-labeled acetylated (K) peptides and 10 TMT-labeled
phosphopeptides were identified and quantified without prior enrichment.
Further experiments include the enrichment of these post-translational modified
peptides prior to TMT-labeling and nanoLC-MS3 analysis for quantitative
analysis.
References
1. Dayon, L. et al. Relative Quantification of Proteins in Human Cerebrospinal
Fluids by MS/MS Using 6-plex Isobaric Tags. Anal. Chem., 2008, 80, 2921-2931.
2. U. De Marchi et al., Influences of Reactive Oxygen Species Production by
Monoamine oxidase activity on aluminium-induced mitochondrial permeability
transition, Cellular and Molecular Life Sciences, 2004, 61, 2664-2671.
3. Ting et al. MS3 Eliminates Ratio Distortion in Isobaric Multiplexed Quantitative
Proteomics. Nature Methods, 2011, 8(11), 937-940.
4. Viner et al. ASMS 2013 poster: Increasing the Multiplexing of Protein
Quantitation from 6-to 10-Plex Reporter Ion Isotopologues.
5. Rigbolt, K.T. et al., GProX, a User-Friendly Platform for Bioinformatics Analysis
and Visualization of Quantitative Proteomics Platform Data, Mol. Cell.
Proteomics, 2011, 10(8):O110.007450.
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This information is not intended to encourage use of these products in any manners that might infringe the
intellectual property rights of others.
PO64105-EN 0614S
6 Liver Mitochondria Proteomics: Protein and PTM Quantitation
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